Research ArticleGlucagon-Like Peptide-1 Receptor Agonist Protects Dorsal RootGanglion Neurons against Oxidative Insult

Mohammad Sarif Mohiuddin,1 Tatsuhito Himeno ,1 Rieko Inoue,1 Emiri Miura-Yura,1

Yuichiro Yamada,1 Hiromi Nakai-Shimoda,1 Saeko Asano,1 Makoto Kato,1 Mikio Motegi,1

Masaki Kondo,1 Yusuke Seino,2 Shin Tsunekawa,1 Yoshiro Kato,1 Atsushi Suzuki,2

Keiko Naruse ,3 Koichi Kato,4 Jiro Nakamura,1 and Hideki Kamiya 1

1Division of Diabetes, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Japan2Division of Endocrinology andMetabolism, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake,Aichi, Japan3Department of Internal Medicine, Aichi Gakuin University School of Dentistry, Nagoya, Japan4Department of Medicine, Aichi Gakuin University School of Pharmacy, Nagoya, Japan

Correspondence should be addressed to Hideki Kamiya; hkamiya@aichi-med-u.ac.jp

Received 15 August 2018; Revised 23 November 2018; Accepted 30 December 2018; Published 21 February 2019

Guest Editor: Julia M. dos Santos

Copyright © 2019 Mohammad Sarif Mohiuddin et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original workis properly cited.

Objective. Diabetic polyneuropathy (DPN) is one of the most prevalent diabetic complications. We previously demonstrated thatexendin-4 (Ex4), a glucagon-like peptide-1 receptor agonist (GLP-1RA), has beneficial effects in animal models of DPN. Wehypothesized that GLP-1 signaling would protect neurons of the peripheral nervous system from oxidative insult in DPN. Here,the therapeutic potential of GLP-1RAs on DPN was investigated in depth using the cellular oxidative insult model applied to thedorsal root ganglion (DRG) neuronal cell line. Research Design and Methods. Immortalized DRG neuronal 50B11 cells werecultured with and without hydrogen peroxide in the presence or absence of Ex4 or GLP-1(7-37). Cytotoxicity and viability weredetermined using a lactate dehydrogenase assay and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt), respectively. Antioxidant enzyme activity was evaluated using a superoxidedismutase assay. Alteration of neuronal characteristics of 50B11 cells induced by GLP-1RAs was evaluated withimmunocytochemistry utilizing antibodies for transient receptor potential vanilloid subfamily member 1, substance P, andcalcitonin gene-related peptide. Cell proliferation and apoptosis were also examined by ethynyl deoxyuridine incorporationassay and APOPercentage dye, respectively. The neurite projection ratio induced by treatment with GLP-1RAs was counted.Intracellular activation of adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling was also quantified aftertreatment with GLP-1RAs. Results. Neither Ex4 nor GLP-1(7-37) demonstrated cytotoxicity in the cells. An MTS assayrevealed that GLP-1RAs amended impaired cell viability induced by oxidative insult in 50B11 cells. GLP-1RAs activatedsuperoxide dismutase. GLP-1RAs induced no alteration of the distribution pattern in neuronal markers. Ex4 rescued the cellsfrom oxidative insult-induced apoptosis. GLP-1RAs suppressed proliferation and promoted neurite projections. NoGLP-1RAs induced an accumulation of cAMP. Conclusions. Our findings indicate that GLP-1RAs have neuroprotectivepotential which is achieved by their direct actions on DRG neurons. Beneficial effects of GLP-1RAs on DPN could be relatedto these direct actions on DRG neurons.

1. Introduction

Among many significant diabetic complications, diabeticpolyneuropathy (DPN) is one of the most prevalent

complications and causes nontraumatic amputations oflower limbs [1]. Due to the lack of therapies to address theetiology of neurodegeneration in the peripheral nervous sys-tem (PNS) of diabetic patients, glucose-lowering therapy is

HindawiJournal of Diabetes ResearchVolume 2019, Article ID 9426014, 10 pageshttps://doi.org/10.1155/2019/9426014

the only effective therapy to prevent the onset and progres-sion of DPN [2]. In the current study, we investigated thebeneficial effects of glucagon-like peptide-1 (GLP-1) signal-ing in neurons of the PNS using an in vitro model of DPN.

GLP-1, an incretin hormone which lowers blood glucoselevels through enhancement of glucose-stimulated insulinsecretion (GSIS), also has pleiotropic effects. In nervous sys-tems, GLP-1 has a regulatory effect on food intake throughthe intermediary of the vagus nerve and the central nervoussystem (CNS) [3–7]. It is known that GLP-1 activates adenyl-ate cyclase and employs cAMP as a second messenger toenhance GSIS in pancreatic beta cells [8, 9]. The cAMP sig-naling has been proven to stimulate neurite outgrowth [10,11] and antagonize apoptosis of PNS neurons or PC12 cells[12]. In some kinds of nonneural cells including pancreaticbeta cells and cardiomyocytes, antiapoptotic effects ofGLP-1 receptor agonists (GLP-1RAs) have been also shown[13–16]. Additionally, it has been reported that activationof GLP-1 signaling modified cell fate and differentiation inpancreatic beta cells [17, 18]. GLP-1 signaling inducedin vivo reprogramming of pancreatic exocrine cells into betacells [17] and in vitro differentiation of human embryonicstem cells into insulin-producing cells [19].

Previously, we reported the beneficial effects of exendin-4(Ex4) (also known as exenatide), a GLP-1RA, in the PNS ofdiabetic mice [20]. In that prior study, we indicated theimprovement of DPN using an in vivo model but the mech-anism of the favorable effects on the PNS has not yet beenidentified. Although we have proven that the elongation ofneurite outgrowth using a tissue culture system of mousedorsal root ganglion (DRG) was accelerated by supplementa-tion of Ex4 or GLP-1, detailed effects of GLP-1RAs in theDRG should be still elucidated.

Among various mechanisms of pathogenesis in DPN,chronic inflammation followed by oxidative stress hasbeen highlighted by several researchers [21, 22]. Forinstance, cyclooxygenase-2-deficient mice were protectedfrom dysfunction of the PNS in experimental diabetes[23]. Given that oxidative stress due to various biologicalpathways, including chronic low-grade inflammation, hasbeen suggested as a pathogenesis and a therapeutic targetof DPN [21, 24, 25], we attempted to provide oxidativestress in our culture system. However, it remains to beclarified which factor is crucial in the pathology of DPN,e.g., glucotoxicity, insulin resistance, or lipotoxicity [21].Therefore, we provided oxidative insult by hydrogen per-oxide, which is a widely used oxidant in experimental set-tings and converts into the stronger oxidant hydroxylradical, in the cell culture system of the DRG neuron cellline to reproduce DPN pathology in this study.

2. Materials and Methods

Unless noted otherwise, all reagents and materials were pur-chased from Thermo Fisher Scientific (Waltham, MA, USA).

2.1. Cell Culture. The DRG neuronal cell line (50B11) estab-lished and kindly provided by Dr. A. Höke (Johns HopkinsUniversity, Baltimore, MD, USA) [26] was incubated at

37°C under 5% CO2 in media consisting of Neurobasal™medium supplemented with 5% fetal bovine serum,2mML-glutamine, and B-27 supplement. 50B11 cells werekept in uncoated plastic tissue culture dishes and regularlypassaged once a week with a 1 : 10-1 : 20 split ratio. For eachexperiment as described in the sections, cells were treatedwith Ex4 (0.1 nM, 1nM, 10 nM, and 100nM), humanGLP-1(7-37) (1 nM, 10nM), or 10μM forskolin. Oxidativeinsult was induced by hydrogen peroxide (0.01mM,0.05mM, and 0.1mM).

2.2. Cell Cytotoxicity Assay. Cells were seeded into 96-wellplates at a density of 1 × 104 cells/well in 100μl medium. Cellcytotoxicity was assessed using lactate dehydrogenase (LDH)assay (Cytotoxicity LDH Assay Kit-WST, Dojindo Laborato-ries, Mashiki, Japan) following the manufacturer’s instruc-tions. The absorbance at 490nm was measured on amicroplate reader (VersaMax, Molecular Devices, Sunnyvale,CA, USA). Cytotoxicity was calculated by the following for-mula: cytotoxicity % = sampleOD – low control OD /high control OD – low control OD × 100 (OD: optical den-sity). Each OD value was calculated by subtracting the back-ground value from each absorbance value.

2.3. Immunocytochemistry. To exclude the possibility of alter-ation in neuronal characteristics by GLP-1RAs which mightinduce a reprogramming of cell fate, the characteristics as asensory neuronal cell were evaluated with the distributionof neuronal markers: transient receptor potential vanilloidsubfamily member 1 (TRPV1), substance P, and calcitoningene-related peptide (CGRP). After a 36-hour culture withor without 100nM Ex4 or 10 nM GLP-1, DRG cells werefixed with 4% paraformaldehyde for 15 minutes. The cellswere blocked with 1% bovine serum albumin, and the follow-ing primary antibodies were applied at 4°C overnight: rabbitpolyclonal anti-TRPV1 antibody (1 : 200; Neuromics, North-field, MN, USA), goat polyclonal anti-substance P antibody(1 : 200; Santa Cruz, Santa Cruz, CA, USA), and goat poly-clonal anti-CGRP antibody (1 : 200; Santa Cruz). After wash-ing, the following secondary antibodies were loaded for 1hour at room temperature in a dark box: Alexa Fluor™594-coupled goat anti-rabbit IgG antibody (1 : 500) or AlexaFluor™ 488-coupled donkey anti-goat antibody (1 : 500).Images were captured by a charge-coupled device (CCD)camera using a fluorescence microscope (IX73, OlympusOptical, Tokyo, Japan).

2.4. Cell Viability Assay. To elucidate the effects of GLP-1RAsin DRG neurons under oxidative stress, cell viability of DRGneurons cultured with or without hydrogen peroxide in thepresence or absence of GLP-1RAs was assessed. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, whichcorrelated mitochondrial activity, was employed to measurecell viability in DRG neurons. Cells were seeded into 96-wellplates at a density of 1 × 104 cells/well in 100μl medium. Cellviability was determined 24 hours after treatment using theCellTiter96™AQueous One Solution Cell Proliferation Assay(Promega Corporation,Madison,WI, USA), which employed

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tetrazolium compoundMTS, according to themanufacturer’sprotocol. The absorbance at 490nmwasmeasured on amicro-plate reader (VersaMax).

2.5. Superoxide Dismutase- (SOD-) Like Activity. To evaluateantioxidant activity, SOD-like activity was measured usingan SOD-like assay kit (Dojindo Inc., Kumamoto, Japan)according to the manufacturer’s instructions [27]. Equalamounts of protein, as determined using a bicinchoninicacid protein assay (Wako Pure Chemical Inc., Osaka, Japan),were applied. Cells were seeded into 96-well plates at a den-sity of 1 × 104 cells/well in 100μl medium. After 24hours,cells were supplemented with GLP-1RAs (10 nM GLP-1,100nM Ex4) or left untouched. After 12 hours of treatmentwith/without GLP-1RAs, the media were replaced withmedia containing 0.1mM hydrogen peroxide. SOD-likeactivity was determined 30 minutes after the exposure withhydrogen peroxide.

2.6. Apoptosis Assay. For the apoptosis assay, 50B11 cellswere seeded into 24-well plates at a density of 5 × 104 cells/-well. Apoptosis was induced by 0.1mM hydrogen peroxide.The degree of apoptosis was assessed using the APOPercen-tage assay (Biocolor, Belfast, Northern Ireland, UK), whichwas performed according to the manufacturer’s instructions.The APOPercentage assay is a dye uptake assay, which stainsonly the apoptotic cells with a purple dye [28]. Apoptoticcells were assessed after a 3-hour exposure to hydrogen per-oxide with or without GLP-1RAs (GLP-1, Ex4) and forskolin.Absorption was measured at 550nm using a microplatereader (VersaMax).

2.7. Cell Proliferation Assay. An ethynyl deoxyuridine (EdU)incorporation assay was performed using the Click-iT PlusEdU Proliferation Kit (Life Technologies Inc., Gaithersburg,MD). Cells were treated with 10μM EdU for 24 hours, thenharvested, and fixed with 4% paraformaldehyde for 20minutes. For EdU detection, cells were incubated withAlexa Fluor™ 488 Azide for 15 minutes and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) [29,30]. The rate of proliferating cells was determined by thenumber of EdU-incorporating cells divided by that ofDAPI-positive cells.

2.8. Neurite Outgrowth Assay in 50B11 Cells. As it has beenverified that the 50B11 neuronal cell line can elongate neur-ites by stimulation with forskolin, the neurite outgrowthinduced by GLP-1RAs was also examined to afford collateralevidence of the neuroregenerative ability in DRG neurons.50B11 cells were plated into 6-well plates at a density of 1× 104 cells/well. Twenty-four hours after the passage of thecells, cells were unexposed or exposed to the indicated com-pounds for 24 h. Images of the cells were captured by acontrast-phase microscope equipped with a CCD cameraand counted for neurite outgrowth which was defined as aprocess equal to or greater than cell bodies in length [31].

2.9. Cyclic Adenosine Monophosphate (cAMP) Assay. CellularcAMP production was measured using an enzyme immuno-assay kit (Cayman Chemical, Ann Arbor, MI, USA) [32, 33].

Cells were seeded into 6-well plates at a density of 5 × 105cells/well. The media were aspirated 20 or 120 minutesafter exposure to test substances, and 250μl of 0.1N HClwas introduced. After 20 minutes incubation at roomtemperature, cells were scraped and centrifuged. Thesupernatants were stored at -80°C until the time of mea-surement. For the experiment with 120-minute exposureto test substances, the medium contained 0.5mM 3-isobu-tyl-1-methyl xanthine (IBMX), a phosphodiesterase inhibi-tor, to inhibit cAMP degradation.

2.10. Statistical Analysis. All the group values were expressedas means ± standard deviation. Data are representative of atleast three independent experiments. The normality of distri-bution was tested by the Kolmogorov-Smirnov test using Rversion 3.4.3 (http://www.r-project.org/, Vienna, Austria,).Statistical analyses were made by Student’s t-test or one-way ANOVA with the Bonferroni correction for multiplecomparisons using StatView version 5.0 (SAS Institute, Cary,NC). The threshold of statistical significance was taken as avalue of p < 0 05. All analyses were performed by personnelunaware of the identities of culture conditions.

3. Results

3.1. No Cytotoxicity Was Introduced by GLP-1RAs in DRGNeurons. There was no significant cytotoxicity induced after24 hour exposure to Ex4 (0.1mM, 1nM, 10nM, or 100 nM)or GLP-1 (1 nM, 10 nM) (absorbance at 490nm: control0 449 ± 0 023, 0.1 nM Ex4 0 414 ± 0 027, 1 nM Ex4 0 355± 0 020, 10 nM Ex4 0 433 ± 0 129, 100 nM Ex4 0 444 ±0 034, 1 nM GLP-1 0 408 ± 0 064, and 10 nM GLP-1 0 424± 0 046) (Figure 1). Neurons were also exposed to an ade-nylate cyclase activator, forskolin. The treatment with10μM forskolin did not induce any significant differencein cytotoxicity (10μM forskolin 0 371 ± 0 029).

3.2. Sensory Neuronal Characteristics in Protein MarkerExpressions Were Not Affected by GLP-1RAs. Ex4 or GLP-1(data not shown) induced no evident changes in the distribu-tion pattern of these sensory neuronal markers comparedwith neurons without those treatments (Figure 2).

3.3. Cell Viability Was Enhanced in DRG Neurons Culturedwith GLP-1RAs. The cell viability of DRG neurons treatedwith 0.1mM hydrogen peroxide for 4 hours was signifi-cantly decreased compared with that of cells cultured withno hydrogen peroxide (control 100 ± 8 1%, 0.1mM hydro-gen peroxide 54 3 ± 2 1, p < 0 01) (Figure 3). However, thetreatment with Ex4 or GLP-1 significantly ameliorated cellviability compared with cells with no treatment (0.1 nMEx4 85 1 ± 13 3, 1 nM Ex4 86 0 ± 6 4, 10 nM Ex4 86 9 ±6 5, 100 nM Ex4 87 5 ± 3 2, 1 nM GLP-1 94 3 ± 11 7, and10 nM GLP-1 92 6 ± 2 9). The supplementation with10μM forskolin also inhibited the decrease of cell viability(84 5 ± 2 6, p < 0 005).

3.4. SOD-Like Activity Increased in the Sensory NeuronsSupplemented with GLP-1RAs. Following exposure to oxida-tive insult with hydrogen peroxide, SOD-like activity

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Figure 1: Cell cytotoxicity of GLP-1 receptor agonists (GLP-1RAs) in dorsal root ganglion (DRG) neurons. Cytotoxicity was determined 24hours after treatment with GLP-1RAs or forskolin using LDH assay. No significant difference was detected between neurons treated withGLP-1RAs or forskolin and those without treatment (control). Concentrations of GLP-1RAs; exendin-4: 0.1, 1, 10, and 100 nM; GLP-1: 1,10 nM. Ctrl: control; Fskln: forskolin; error bar: standard deviation. n = 3 in each group.

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Figure 2: Distribution of sensory neuronal markers in the dorsal root ganglion (DRG) neuron cell line treated with exendin-4. Pictures on theleft side are neurons without any treatment. Pictures on the right side are neurons treated with 100 nM exendin-4 for 36 hours. TRPV1: red(a), substance P: green (b), CGRP: green: DAPI (c), scale 100μm.

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increased in neurons supplemented with GLP-1 or Ex4(cells with no hydrogen peroxide 40 4 ± 6 7%, 10 nMGLP-1 with 0.1mM hydrogen peroxide 54 3 ± 5 8, and100nM Ex4 with 0.1mM hydrogen peroxide 59 9 ± 8 4, p< 0 001 versus cells with no hydrogen peroxide in eachGLP-1RA-supplemented group) (Figure 4).

3.5. Apoptosis Was Prevented in the Neurons Supplementedwith Ex4. Apoptosis evoked by 0.1mM hydrogen peroxidewas detected using the APOPercentage assay (Figure 5).The degree of apoptosis was significantly decreased in theneurons supplemented with 100nM Ex4 (absorbance at550nm: control 0 304 ± 0 017, 100 nM Ex4 0 250 ± 0 014,p < 0 0001) and 10μM forskolin (0 199 ± 0 016, p < 0 0001).However, GLP-1 produced no significant change in the apo-ptosis assay (0 299 ± 0 03, p = 0 623).

3.6. Cell Proliferation Was Suppressed by GLP-1RAs. TheEdU incorporation assay revealed a decrease of proliferationrate of neurons cultured with 10 nM GLP-1 or 100nM Ex4(control 87 7%±5 6%, GLP-1 75 5 ± 10 4, and Ex4 74 1 ±14 4) (Figure 6). However, forskolin had no significant effecton the proliferation rate (forskolin: 86 9 ± 6 2).

3.7. Neurite Outgrowth Was Induced with GLP-1RAs. Thepercentage of neurons with neurite(s) increased in the neu-rons cultured with Ex4 or GLP-1 compared with the control(control 8 7%±5 1%, 100 nM Ex4 28 2 ± 4 0, and 10 nMGLP-1 23 3 ± 6 5, p < 0 0001 for both cases versus control)(Figure 7).

3.8. The Adenylate Cyclase/cAMP Pathway Was NotActivated by GLP-1RAs in DRG Neurons. Cyclic AMP levelsafter stimulation with GLP-1RAs and forskolin weredetermined. After 20 minutes of stimulation with 10μM for-skolin, cAMP had accumulated in the neurons (control: 5 3± 0 3 pmol/ml, 10μM forskolin: 234 5 ± 6 3, p < 0 0001)(Figure 8). However, no accumulation of cAMP was detectedin the neurons treated with Ex4 and GLP-1 (10 nM GLP-1:3 3 ± 0 4, 100 nM Ex4: 4 0 ± 0 4). Longer exposure to GLP-1RAs supplemented with a phosphodiesterase inhibitor alsogenerated no significant cAMP accumulation (Supplementalfigure available here).

4. Discussion

In this decade, drug development targeting GLP-1 signalinghas been considered as a prospective therapy of type 2 diabe-tes. A novel GLP-1RA semaglutide which can be orallyadministered would accelerate popularization of GLP-1RAsin clinical settings [34]. Furthermore, the neuroprotectiveeffects of Ex4 have been already proven in one clinical trialof Parkinson’s disease [35]. Therefore, if the neuroprotectiveeffects of GLP-1RAs are accepted amongst the scientific com-munity, a drug repositioning strategy of GLP-1RAs targetingother diseases will be promising, especially in diabetic com-plications including DPN.

In the current study, we investigated the neuroprotectiveeffects of GLP-1RAs in the DRG neuronal cell line. First, weexamined the neurotoxicity of GLP-1RAs in the DRG neu-rons. Second, we examined the effect of GLP-1RA on cell

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Figure 3: Cell viability in dorsal root ganglion (DRG) neurons treated with GLP-1 receptor agonists. Cell viability was quantified using3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS). Although hydrogenperoxide (H2O2) significantly decreased the cell viability of DRG neurons, GLP-1 receptor agonists, exendin-4 and GLP-1, and forskolin,an activator of adenylate cyclase, prevent the reduction of cell viability induced by H2O2. White bar: no supplementation of H2O2; hatchedbar: 0.01mM H2O2; filled bar: 0.1mM H2O2; Ctrl: control; H2O2: hydrogen peroxide; #p < 0 01 versus control without H2O2;

∗p < 0 05versus control with 0.1mM H2O2;

∗∗p < 0 005 versus control with 0.1mM H2O2; error bar: standard deviation. n = 3 in each group.

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viability, antioxidant enzyme activity, and apoptosis in theDRG neurons. We confirmed enhanced cell viability,increased activity of antioxidant enzyme SOD, and inhibitionof apoptosis with GLP-1RA supplementation. We then dem-onstrated that treatment with GLP-1RAs reduced cell prolif-eration and promoted neurite outgrowth of DRG neurons.Although these significant changes were seemed to be evoked

by activation of the adenylate cyclase/cAMP pathway, no evi-dent accumulation of intracellular cAMP was generated bystimuli with GLP-1RAs.

GLP-1RAs have previously been shown to promote neur-ite outgrowth in PC12 cells, a rat pheochromocytoma celltype [36, 37]. However, no report has investigated the directpharmacological function of GLP-1RAs in the cells of thePNS, e.g. DRG neurons, Schwann cells, vascular endothelialcells in peripheral nerves. Some research studies, includingour previous study, have already reported in vivo beneficialeffects of GLP-1RAs in the disorders of the PNS [20, 38].The current study would support these beneficial effectsthrough verification of the direct effects of GLP-1RAs onDRG neurons.

A number of DPN pathogenesis mechanisms have beenpostulated in experimental studies, including the polyolpathway, advanced glycation end products, poly ADP-ribose polymerase, the protein kinase C pathway, and oxida-tive stress [39, 40]. In the current study, we chose oxidativestress to represent an in vitroDPNmodel. To verify the novelin vitro experimental system for investigation of DPN, weconfirmed the characteristics of a 50B11 cell line as DRGneurons and induced oxidative insult on the cell line. Afterthe confirmation of no cytotoxicity of GLP-1RAs and forsko-lin in 50B11, we evaluated the neuronal characteristics of thecells. The markers of a primary sensory neuron includingTRPV1, substance P, and CGRP were expressed in 50B11even after the treatment with GLP-1RAs. Furthermore, wesuccessfully performed the neurite outgrowth assay, whichis accepted as one of the crucial neuronal assays in asympathetic-like neuron cell line PC12 [31]. As oxidativestress is one of the primary factors according to the prevailingviews of DPN pathogenesis [39], we attempted to producethe pathogenesis utilizing hydrogen peroxide in the neuronal

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Figure 4: Superoxide dismutase- (SOD-) like activity in dorsalroot ganglion (DRG) neurons treated with glucagon-like peptide-1 (GLP-1) receptor agonists. Oxidative insult induced by 30-minute treatment with 0.1mM hydrogen peroxide increasedSOD-like activity in the neurons supplemented with 10 nM GLP-1 or 100 nM exendin-4. ∗p < 0 001 versus no treated cell withH2O2. H2O2: hydrogen peroxide; Ctrl: control; GLP-1: cellssupplemented with 10 nM GLP-1; Ex-4: cells supplemented with100 nM exendin-4. Error bar means standard deviation. n = 6 or7 in each group.

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Figure 5: Apoptosis in dorsal root ganglion (DRG) neurons treatedwith exendin-4. Apoptosis induced by 3-hour treatment with0.1mM hydrogen peroxide was partially inhibited in theneurons supplemented with 100 nM exendin-4 or 10μM forskolin.∗p < 0 05 versus control; H2O2: hydrogen peroxide; PC: positivecontrol of apoptosis; GLP-1: glucagon-like peptide-1; Ex-4:exendin-4. Error bar means standard deviation. n = 8 in each group.

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Figure 6: Proliferation rate of dorsal root ganglion (DRG) neuronstreated with GLP-1 receptor agonists. Proliferation rate assessed byEdU assay revealed that both GLP-1 receptor agonists, exendin-4and GLP-1, suppressed proliferation of DRG neurons. Ctrl:control; Ex-4: cells supplemented with 100 nM exendin-4; GLP-1:cells supplemented with 10 nM GLP-1; ∗p < 0 05 versus control;error bar: standard deviation. n = 9 in each group.

6 Journal of Diabetes Research

cell culture. Although, in clinical settings, several factorsincluding dyslipidemia, hyperglycemia, hypertension, andsmoking are considered to be risk factors of DPN [41], the

significance of each oxidation mechanism derived from glu-cose, proteins, or lipids is unclear in the pathogenesis ofDPN. Therefore, we utilized hydrogen peroxide, which isconsidered to be one of the most important reactive oxygenspecies because it crosses membranes and yields hydroxylradicals via Fenton reaction in cells [42], as an oxidativeinsult-mimicking oxidative stress in DPN. As a result, hydro-gen peroxide provoked an increase of antioxidant SOD in50B11 cells. These experiments verified our experimentalsystem as a novel approach to investigate DPN.

However, we must recognize some limitations of ourstudy. As it is known that the incretin/adenylate cyclase/-cAMP pathway is critical for insulin secretion in pancreaticbeta cells [43] and neuroprotective effect in the CNS neurons[9], we compared pharmacological effects of GLP-1RAs withthose of forskolin, an activator of adenylate cyclase, in DRGneurons. We proved the antiapoptotic effect of Ex4 and for-skolin and the decrease of cell proliferation by GLP-1RAs.These findings were consistent with the previous report inwhich liraglutide, another GLP-1RA, potentiated cell viabilityandprevented apoptosis via cAMPsignaling in SH-SY5Yneu-roblastoma cells [44]. Furthermore, neurite outgrowth wasinduced by GLP-1RAs and forskolin. Given that background,these changes appear to indicate the activation of intracellularadenylate cyclase/cAMPsignalingbyGLP-1RAs aswell as for-skolin. However, unexpectedly, cAMP accumulation was notevident in the neurons cultured with GLP-1RAs for 20 or120 minutes. This unexpected finding could be caused by theexperimental limitation that our cAMPmeasurement kit wasable to examine only the endpoint accumulation of cAMP.The activation of adenylate cyclase induced by GLP-1RAsmight be more transient than we expected. Therefore, in thefuture,wewould like tomeasure cAMPaccumulationutilizinga real-time detection system.

Furthermore, we should consider scrutinizing other sig-naling pathways which have been reported to be initiatedby GLP-1RAs. It is known that p44/42 mitogen-activatedprotein kinase (also called ERK1/2) can be also activated byGLP-1 in pancreatic beta cells [45]. It is also shown that theantiapoptotic effect of GLP-1 is mediated by ERK1/2 activa-tion in beta cells [46]. Therefore, the antiapoptotic effectshown in the current study might be mediated by activationof ERK1/2 signaling.

Another limitation is the immortalization of the neurons.As the DRG neuronal cell line 50B11 cells are immortalizedneurons, the differences between nonproliferative neuronscollected from mammalians and the genetically engineeredneurons should be taken into account. It was reported thatan activation of phosphoinositide-3-kinase (PI3K) inducedby GLP-1 in the beta cell line accelerated mitosis of the cells[47]. However, in this study, EdU incorporation wasdecreased by administration of GLP-1RAs. To address thisconflict, in the future, we would clarify the involvement ofPI3K signaling in sensory neurons [45, 46, 48].

5. Conclusions

This study is the first report to investigate the neuroprotec-tive effects of GLP-1RAs on DRG neurons. The beneficial

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Figure 7: Neurite outgrowth of dorsal root ganglion (DRG)neurons. The ratio of neurite-positive neurons increased in cellssupplemented with GLP-1 receptor agonists, exendin-4 and GLP-1, as well as cells which were supplemented with forskolin. Ctrl:control; Ex-4: cells supplemented with 100 nM exendin-4; GLP-1:cells supplemented with 10 nMGLP-1; forskolin: cells supplementedwith 10 nM forskolin; ∗∗p < 0 001 versus control; error bar: standarddeviation.n = 9or15ineachgroup.

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mol

/ml)

Figure 8: Intracellular cyclic adenylate monophosphate (cAMP)accumulation in neurons treated with GLP-1 receptor agonists.The cAMP accumulation was measured 20 minutes after exposureto 100 nM exendin-4, 10 nM GLP-1, or 10 μM forskolin. BothGLP-1 receptor agonists, exendin-4 and GLP-1, provoked nosignificant cAMP accumulation. Ex-4: cells supplemented with100 nM exendin-4; ∗∗p < 0 001 versus control; error bar: standarddeviation. n = 5 or 6 in each group.

7Journal of Diabetes Research

effects of GLP-1RAs in DPN might be attributable to thedirect neuroprotective effects of GLP-1RAs on DRG neuronsthrough protection from cellular oxidative insult.

At the same time, we successfully verified the novelin vitro experimental system for investigation of DPN.

Abbreviations

cAMP: Cyclic adenosine monophosphateCCD: Charge-coupled deviceCGRP: Calcitonin gene-related peptideCNS: Central nervous systemDAPI: 4′,6-Diamidino-2-phenylindoleDRG: Dorsal root ganglionDPN: Diabetic polyneuropathyEdU: Ethynyl deoxyuridineEx4: Exendin-4GLP-1: Glucagon-like peptide-1GLP-1RA: GLP-1 receptor agonistGSIS: Glucose-stimulated insulin secretionIBMX: 3-Isobutyl-1-methyl xanthineLDH: Lactate dehydrogenaseMTS: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt

PNS: Peripheral nervous systemSOD: Superoxide dismutaseTRPV1: Transient receptor potential vanilloid subfamily

member 1.

Data Availability

The whole data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this paper.

Acknowledgments

This research was supported in part by the Grant-in-Aidfor Scientific Research (15H06720) from the Ministry ofEducation, Culture, Sports, Science and Technology(MEXT) and Grants for Young Researchers from the JapanAssociation for Diabetes Education and Care. TH was sup-ported by the Manpei Suzuki Diabetes Foundation, JapanDiabetes Foundation, and Aichi Medical University Aikei-kai. The authors wish to acknowledge helpful commentsand discussions from Prof. Eva L. Feldman. The authorswould like to thank Prof. A. Höke for kindly providing50B11 neuronal cells and Crystal Pacut, Carey Backus,and John M. Hayes for preparing the cell culture systemusing the 50B11 neuronal cell line.

Supplementary Materials

Supplemental figure: intracellular cyclic adenylate monopho-sphate (cAMP) accumulation in neurons treated with GLP-1

receptor agonists and a cAMP/cGMP-phosphodiesteraseinhibitor. (Supplementary Materials)

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